In recent years, genetic information of many organisms, such as human genome, has been decoded. Under the circumstances, functional analysis of proteins and creation of genomic medicine based on such genetic information have been attracting attention for postgenomic studies. Application and utilization of proteins corresponding to such genetic information for pharmaceutical products and the like requires easy synthesis of extensive kinds of proteins in a short time.
At present, expression systems using viable cells (hereinafter sometimes to be referred to as “cell-system”) of yeast, insect cell and the like by the gene recombination technique have been widely utilized as the production methods of proteins. However, viable cells show a propensity toward elimination of exogenous proteins for their functional retention, and there are many proteins that cannot be expressed easily since expression of cytotoxic proteins in viable cells prevents cell growth.
On the other hand, as a production method of protein free of a cell-system, cell-free protein synthesis has been known, which includes adding a substrate, an enzyme and the like to a cell rupture, extract solution and the like to provide a wide choice of genetic information translation systems of organisms in test tubes, and reconstructing a synthetic system capable of linking the necessary number of amino acid residues in a desired order using an mRNA encoding an object protein. Such a cell-free protein synthesis is relatively free of the limitation imposed on the above-mentioned cell-system protein synthesis, and is capable of synthesizing proteins without killing the organism. In addition, because the production of protein does not accompany operations of culture and the like, the protein can be synthesized in a short time as compared to cell-systems. Moreover, inasmuch as the cell-free protein synthesis also affords a large scale production of proteins consisting of amino acid sequences not utilized by the organism, it is expected to be a promising expression method. As a cell rupture or extract solution to be applied to the cell-free protein synthesis, use of various substances of biological derivation has been considered and investigations are underway. Of such substances, since insect cells do not require, unlike many mammalian culture cells, culture under a carbon dioxide atmosphere, can be cultured in a serum-free medium, and can express in a large amount in a cell-system while retaining the inherent biological activity with posttranslational modification, they are used for the expression of various proteins. If the insect cell can be used for a cell-free system, posttranslational modification, such as glycosylation and the like, is fully expected to be applicable. Thus, the development of utilization of the insect cell is drawing attention.
Conventionally, for extraction from an insect cell for the preparation of an extract solution for cell-free protein synthesis, a method comprising reducing the pressure applied to an insect cell after pressurization in an inert gas atmosphere, thereby to rupture the insect cell to allow for extraction, is known (e.g., JP-A-2000-325076). This method nevertheless requires special devices and tools for extraction from the insect cell, and the operation is complicated. Moreover, complicated manipulations are necessary for the setting of the conditions, because protein synthesis ability of the cell-free system vastly change depending on the number of cells, nitrogen gas pressure and pressurization time during the preparation of the extract solution. In addition, the amount of protein synthesized using the extract solution obtained by this method is extremely small, which is of the level that can be measured by the uptake of the radiolabeled amino acid.
Therefore, the development of an insect cell-derived extract solution, which is easy to prepare and which affords synthesis of a large amount of protein, and the preparation method thereof is desired.